Alpha-1 antitrypsin (AAT) deficiency, due to the piZZ mutation, results in life-threatening lung and liver diseases in children and adults. Currently, gene addition therapy is being tested to prevent lung disease. However, this strategy does not halt liver disease progression due to the accumulation of mutant PiZZ protein in the endoplasmic reticulum of liver cells rather than normal secretion into the blood and body fluids. From our previous two year R01 application funded from the ARRA (R01DK084033), we have generated a substantial dataset, demonstrating that : 1) AAV vectors can efficiently deliver self-complimentary shRNA to knock down piZZ AAT expression at both mRNA and protein levels, resulting in improved liver pathology of piZZ mice. 2) Alternative combinatorial approaches can be used to both knockdown piZZ expression and restore functional, circulating AAT; These approaches include liver AAV-shRNA delivery followed by muscular injection of an AAV-wtAAT cassette, dual vector administration into the liver with independent vectors for shRNA and an optimized AAT cassette that avoids shRNA-based degradation, and finally delivery of a single vector that simultaneously delivers shRNA and optimized AAT into the liver. The dual-targeting strategy applied in these experiments simultaneously prevents both liver and lung disease development in AAT deficiencies. However, AAV delivery of shRNA faces three obstacles: capsid specific CTL-mediated elimination of AAV-transduced liver cells, potential off-target effects of shRNA in non-targeted, transduced tissue, and the high prevalence of neutralizing antibodies (Nab) to AAV in the human population. In response to these obstacles, the primary objectives of this renewal proposal are to: (i) investigate the effect of AAV capsid specific CTL-mediated killing on AAV-transduced liver cells in piZZ mice after treatment with scAAV/shRNA vector (ii) develop novel approaches to exclusively control shRNA expression in the liver of piZZ mice (iii) generate AAV mutants capable of liver-specific transduction and evasion of neutralizing antibody activity. The long-term goal of the proposal is to design safer and more effective AAV vectors for gene therapy in patients with AAT deficiency. PUBLIC HEALTH RELEVANCE: Adeno-associated virus (AAV) vectors are currently being employed in clinical trials of patients with alpha-one antitrypsin (AAT) deficiency. AAT deficiency clinical manifestations include both liver and lung disease resulting from nonfunctional protein accumulation in liver cells and a lack of circulating AAT, respectively. To prevent the liver disease, we have successfully used AAV vectors to deliver short hairpin RNA and suppress misfolded protein expression. Still, AAV gene therapy application in AAT deficiency faces several key restrictions pertaining to host immune response against the therapeutic vector - thus the proposed studies will carefully examine these immune responses and test several alternative solutions including development of novel AAV virions with improved liver-specific targeting and enhanced evasion from host immunity.